Method for detecting antibody and antigen for detecting Borna disease virus

ABSTRACT

With respect to immunoglobulins that are raised against an exogenous antigen, when the class switching from IgM to IgG necessitates a long period of time, detect of IgM antibody alone, or concurrent detect of the IgM antibodies and IgG antibodies to the exogenous antigen is achieved. An antigen polypeptide for performing the examination of an antibody to Borna disease virus (may be referred to as “BDV”) as an example of such an exogenous antigen in a more accurate manner, and a method for detecting anti-BDV antibody in which such a polypeptide is used are provided.

FIELD OF THE INVENTION

[0001] The present invention relates to a method for detecting anantibody to a exogenous antigen such as Borna disease virus or the like,and an antigen for detecting Borna disease virus.

BACKGROUND

[0002] There exists each class of immunoglobulins such as IgG, IgA, IgM,IgD and IgE. IgM antibodies are first raised in response to thestimulation of an antigen, and rapidly disappear owing to the short halflife thereof. Thus, they are gradually replaced with IgG. Allogenichemagglutinin, rheumatoid factors, heterophile antibodies and coldagglutinin predominantly belong to this IgM (Rinsho Kensaho Teiyou(Guidebook on laboratory test), Kanehara & Co., Ltd., 31st. Edition,1998, p.820).

[0003] In cases of infectious diseases, it is diagnostically useful tomeasure both specific antibody titers of the serum in the initial stageof the infection and of the serum in the stage convalescent, pairwise(Guidebook on laboratory test), Kanehara & Co., Ltd., 31st. Edition,1998, p.808). For example, a method for detecting IgM has been reportedfor the purpose of determining the infection with human cytomegalovirus(HCMV) (Japanese Translation Provisional Publication No.Hei10-502253).However, there has not been disclosed any method for concurrentlydetecting IgG and IgM.

[0004] Class switching from IgM to IgG of immunoglobulins has beengenerally known to be achieved within approximately one month followingthe appearance of the IgM. However, many of the natures of candidatesubstances which may be an antigen have been unknown, and a period oftime required for the class switching of an immunoglobulin as well asother characteristics have not yet been elucidated in many aspects.

[0005] For example, in connection with Borna disease which is an immunerelated neurologic syndrome of which causative virus is Borna diseasevirus (hereinafter, referred to as “BDV”), many matters have not beenelucidated yet. Borna disease is a viral encephalomyelitis caused in ahorse through the four seasons in Germany as well as surrounding nationsthereof. It exhibits symptoms such as cerebral palsy, affective disorderand the like, which may be lethal when it follows the acute process.When a rat is experimentally infected with this virus, a polyphasicsyndrome characterized by hyperkinesia, stereotyped behavior, dyskinesiaand ataxia is developed (O. Narayan et al., Science, 220:1401-1403(1983)).

[0006] BDV has been known of its pathogenicity toward horses that arethe natural hosts thereof, and the existence of an antibody that reactswith BDV was indicated in the serum of a patient suffering from a mentaldisorder in 1985, suggesting the probability of pathogenesis also towardhumans. Infectious epidemiologic studies of BDV shall greatly contributeto understandings of the mental disorders. Recently, a BDV gene has beenfound in hemocytes including gene clusters of mental disorders with acertain frequency.

[0007] Because BDV proliferates at just a low titer, purification forexecuting an analysis is difficult. Diagnoses of BDV infection have beencarried out through detecting the appearance of clinical symptoms thatare common in this disease, and detecting a serum antibody that detectsa viral protein in an infected cell by an indirect immunofluorescencetechnique (IFT) (G. Pauli et al., Zbl. Vet. Med. [B], 31: 552-557(1984)), Western blot, immunoprecipitation or the like. Operation inthese methods is complicated, therefore, it is difficult to use those ina mass investigation of a group of humans or livestocks.

[0008] The sequence of BDV has been already elucidated, and a method fordetecting an anti-BDV antibody by an ELISA method has been reported,where p23, recp23, p40 and recp40 antigens are used (Japanese PatentProvisional Publication No. 2001-190288). In addition, a test methodinvolving the determination of a BDV specific circulating immune complex(CIC) in plasma was also reported, where p40 and p24 antigens have beenused (Japanese Translation Provisional Publication No. 2002-500363).Moreover, a method for detecting an antibody by magnetic beads in whichan antigen polypeptide including a specific amino acid sequence selectedfrom p40 and p24 regions was also reported (Japanese Patent ProvisionalPublication No.Hei11-180998)

[0009] However, characteristic features of BDV have been still far frombeing sufficiently elucidated, and many points remain unknown inconnection with the period when an antibody to BDV is raised or with theaccurate method for detecting an antibody. Accordingly, development ofmore accurate method for detecting an antibody has been desired.

[0010] An object of the present invention is to provide a reagent fordetecting an anti-BDV antibody for more accurately carrying out theexamination of an antibody to a exogenous antigen, particularly BDV, andto provide a method for detecting an anti-BDV antibody in which thereagent is used.

[0011] [Disclosure of the Invention]

[0012] The present inventors elaborately investigated in order toachieve the problems as described above, and focused attention to theexistence of immunoglobulins, which are raised against a exogenousantigen, that may often necessitate two months or longer, morespecifically, one year or longer for the class switching from IgM toIgG. Accordingly, the method for detecting an antibody of the presentinvention was accomplished. In addition, the present inventors foundthat the aforementioned problems can be solved through using an antigenselected from the p10 region alone, or in combination with an antigenpolypeptide selected from the p24 region and/or p40 region, among theproteins that constitute BDV. Thus, the reagent for detecting ananti-BDV antibody of the present invention was accomplished.

[0013] Accordingly, the present invention is directed to:

[0014] 1) A method for detecting an antibody wherein an examination of adisease caused by an exogenous antigen is conducted,

[0015] said exogenous antigen being an antigen having a property inwhich the class switching from the IgM antibody to the IgG antibody ofimmunoglobulin antibodies raised against said antigen is achieved aftertwo months following the appearance of the IgM antibody,

[0016] said method comprising detecting the IgM antibody to saidexogenous antigen;

[0017] 2) The method for detecting an antibody according to the aboveitem 1) wherein the detection of the antibody to said exogenous antigenis carried out by detecting the IgM antibody alone, or concurrentlydetecting the IgM antibody and IgG antibody;

[0018] 3) The method for detecting an antibody according to the aboveitem 1) wherein said exogenous antigen is a microorganism, virus and/orproteinous substance which may be the cause of a disease of a human orof a mammal other than humans;

[0019] 4) The method for detecting an antibody according to the aboveitem 1) wherein said exogenous antigen is Borna disease virus (BDV);

[0020] 5) A method for detecting an antibody wherein an examination of adisease caused by Borna disease virus (BDV) is conducted,

[0021] said method comprising detecting the IgM antibody alone, orconcurrently detecting the IgM antibody and IgG antibody which is(are)raised against the virus;

[0022] 6) The method for detecting an antibody according to the aboveitem 1) wherein said method for detecting an antibody is an immuneagglutination reaction method;

[0023] 7) The method for detecting an antibody according to the aboveitem 6) wherein said immune agglutination reaction method is a fineparticle counting immunoassay method;

[0024] 8) A reagent for detecting an anti-BDV antibody which has anantigen polypeptide selected from the p10 region of a Borna diseasevirus (BDV) protein;

[0025] 9) The reagent for detecting an anti-BDV antibody wherein theantigen polypeptide according to the above item 8) comprises an antigenpolypeptide has at least 8 amino acids;

[0026] 10) The reagent for detecting an anti-BDV antibody wherein theantigen polypeptide according to the above item 8) has a polypeptidewhich includes an amino acid sequence set out in SEQ ID NO: 5, 6, 7 or8;

[0027] 11) A method for detecting an anti-BDV antibody wherein theantigen polypeptide according to the above item 8) is used;

[0028] 12) A method for detecting an anti-BDV antibody wherein theantigen polypeptide according to the above item 8), and an antigenpolypeptide selected from the p24 region and/or p40 region of a Bornadisease virus (BDV) protein are used;

[0029] 13) The method for detecting an anti-BDV antibody wherein theantigen polypeptide from the p24 region according to the above item 12)has a polypeptide including an amino acid sequence set out in SEQ ID NO:1 or 2;

[0030] 14) The method for detecting an anti-BDV antibody wherein theantigen polypeptide from the p40 region according to the above item 12)has a polypeptide including an amino acid sequence set out in SEQ ID NO:3 or 4;

[0031] 15) The method for detecting an anti-BDV antibody according tothe above item 11) wherein said method for detecting an anti-BDVantibody is an immune agglutination reaction method; and

[0032] 16) The method for detecting an antibody according to the aboveitem 15) wherein said immune agglutination reaction method is a fineparticle counting immunoassay method.

BRIEF DESCRIPTION OF DRAWINGS

[0033] [FIG. 1]

[0034] A drawing showing results of the detection of a sample collectedin 1998 of specimen No. 5 (Experimental Example 1).

[0035] [FIG. 2]

[0036] A drawing showing results of the detection of a sample collectedin 1999 of specimen No. 5 (Experimental Example 1).

[0037] [FIG. 3]

[0038] A drawing showing results of the detection of a sample collectedin 2001 of specimen No. 5 (Experimental Example 1).

[0039] [FIG. 4]

[0040] A drawing showing results of the detection of a sample collectedin 1998 of specimen No. 6 (Experimental Example 1).

[0041] [FIG. 5]

[0042] A drawing showing results of the detection of a sample collectedin 1999 of specimen No. 6 (Experimental Example 1).

[0043] [FIG. 6]

[0044] A drawing showing results of the detection of a sample collectedin 2000 of specimen No. 6 (Experimental Example 1).

[0045] [FIG. 7]

[0046] A drawing showing results of the detection of a sample collectedin 2001 of specimen No. 6 (Experimental Example 1).

[0047] [FIG. 8]

[0048] A drawing showing results of the detection of a sample collectedin 1998 of specimen No. 25 (Experimental Example 1).

[0049] [FIG. 9]

[0050] A drawing showing results of the detection of a sample collectedin 1998 of specimen No. 26 (Experimental Example 1).

BEST MODE FOR CARRYING OUT THE INVENTION

[0051] Upon infection with a virus or the like that is an exogenousantigen, IgM first is raised, then an IgG antibody is raised whilegradual decrease of the amount of the IgM. Usually, it is after 2 to 4weeks passed following the infection when the IgG is raised, therefore,examination of the infection with a virus or the like is carried outwith targeting to IgG, in general. However, the fact that a period oftime required for the class switching from IgM to IgG varies dependingupon the nature of the antigen has been scarcely reported heretofore. Asa consequence of the study of a certain type of viruses, particularly, aslight amount of viruses, the present inventors found cases in which theclass switching from IgM to IgG requires one year or longer. In suchinstances, it is probable that early stages of viral infections areoverlooked when IgG alone is measured. Thus, the present inventorsinvestigated method for carrying out the examination of a disease causedby an exogenous antigen in a more accurate manner, which enables themeasurement of an antibody also in the early stage of the infection, inwhich the IgM is detected.

[0052] (Exogenous Antigen)

[0053] According to the present invention, the exogenous antigen refersto a substance that is not a component in a self living body and to anantigen that invades from outside the living body. It should bedistinguished from endogenous antigens which occur due to a tissue ofthe self living body as in the case of autoimmune diseases. Examples ofsuch antigens may be substances which may be the cause of a kind of adisease through invading from the external body without particularlimitation, but for example, they include microorganisms such asbacteria, fungi and the like, viruses and antigenic substances such asproteins.

[0054] Specific examples of the exogenous antigen to which the methodfor detecting an antibody according to the present invention may beapplied include some types of viruses. For example, when the amount ofthe infected virus is slight, and thus isolation of the virus from theliving body is difficult, direct measurement of the viral component isalso difficult. Therefore, examination of the infection with the virusis difficult except for by determining the appearance of the antibody tothe virus. Examples of such viruses include BDV belonging to orderMononegavirales that infects nerve cells and requires a long period oftime for the class switching from IgM to IgG.

[0055] On the other hand, illustrative examples of the virus thatinfects nerve cells, followed by embedding of the virus itself into thenerve cells, with the occurrence of the class switching from IgM to IgGin a short period of time include e.g., viruses belonging to familyHerpesviridae, family Papovaviridae, family Retroviridae as well asmeasles virus. Viruses belonging to family Herpesviridae are classifiedinto subfamily Alphaherpesvirinae, subfamily Betaherpesvirinae,subfamily Gammaherpesvirinae and the like. Specifically, viruses aspresented below are involved.

[0056] Family Herpesviridae

[0057] Subfamily Alphaherpesvirinae

[0058] Genus Simplexvirus

[0059] B virus=simian herpesvirus,

[0060] Bovine thelitis virus=bovine herpesvirus,

[0061] Herpes simplex virus 1,2=human herpesvirus 1,2

[0062] Genus Varicellovirus

[0063] Aujeszky virus=porcine herpesvirus 1

[0064] Infectious bovine rhinotracheitis virus

[0065] (IBR virus)=bovine herpesvirus 1

[0066] Equine abortion herpesvirus=equine herpesvirus 1

[0067] Herpes zoster virus=human herpesvirus 3

[0068] Others

[0069] Feline viral rhinotracheitis virus=feline herpesvirus 1

[0070] Canine trachea bronchitis virus=canine herpesvirus

[0071] Infectious laryngotracheitis virus=avian herpesvirus 1

[0072] Duck plague virus=duck herpesvirus 1

[0073] Bovine encephalitis herpesvirus=bovine herpesvirus 5

[0074] Coital exanthema virus=equine herpesvirus 3

[0075] Subfamily Betaherpesvirinae

[0076] Genus Cytomegalovirus

[0077] Genus Roseolovirus

[0078] Human herpesvirus 6

[0079] Others

[0080] Porcine herpesvirus 2

[0081] Equine herpesvirus 2

[0082] Equine herpesvirus 5

[0083] Equine herpesvirus 7

[0084] Subfamily Gammaherpesvirinae

[0085] Genus Lymphocryptovirus

[0086] Epstein-Barr virus=human herpesvirus 4

[0087] Chimpanzee herpesvirus

[0088] Baboon herpesvirus

[0089] Family Papovaviridae

[0090] Genus Polyomavirus

[0091] Simian renal cell vacuolation virus

[0092] Mouse polyomavirus

[0093] Bovine polyomavirus

[0094] Genus Papillomavirus

[0095] Bovine papillomavirus 1-6

[0096] Canine oral papillomavirus

[0097] Family Retroviridae

[0098] Genus “Mammalian type C retroviruses”

[0099] Mammalian type C viruses

[0100] Feline leukemia virus

[0101] Porcine type C virus

[0102] Genus “BLV-HTLV retroviruses”

[0103] Equine leukemia virus=BLV

[0104] Human T lymphotropic virus 1=HTLV-1

[0105] Human T lymphotropic virus 2=HTLV-2

[0106] Simian T lymphotropic virus

[0107] Genus Lentivirus

[0108] Equine infectious anemia virus

[0109] Bovine immunodeficiency virus

[0110] Feline immunodeficiency virus

[0111] Caprine arthritis encephalitis virus

[0112] Human immunodeficiency virus 1=HIV-1

[0113] Human immunodeficiency virus 2=HIV-2

[0114] Simian immunodeficiency virus=SIV

[0115] Further, the method for the detection of the present inventioncan be applied to any exogenous antigen which can be the cause of adisease of any of humans and mammals other than humans. For example,microorganisms which are zoonotic infectious, specifically, infectiousmicroorganisms toward horse, cattle, sheep, cat, monkey, murine, ostrichor the like are included.

[0116] In addition, the method for the detection of the presentinvention can be suitably applied to exogenous antigens having aproperty in which the class switching from IgM to IgG necessitates along period of time. The period of time required for the class switchingis not particularly limited, however, for example, 2 months or longer,preferably 3 months or longer, more preferably 1 year or longer isrequired, and furthermore, it is suitably applied to antigens thatcomplete the class switching within 10 years, preferably within 7 years,more preferably within 4 years. The present inventors verified theappearance of immunoglobulins that executed class switching from IgM toIgG over a period of about one year in response to BDV infection.Currently, antigens that necessitate such a long period of time forclass switching have been scarcely identified except for BDV, however,it goes without mentioning that when an antigen having such a propertyis newly found, the method for the measurement of the present inventioncan be applied also to such a substance.

[0117] (Antigen Polypeptide)

[0118] The antigen polypeptide of the present invention, i.e., any oneof the antigen polypeptides presented below can be used for diagnosingthe infection with BDV. Specifically, an antigen polypeptide describedin any of the following 1) to 4) can be used.

[0119] 1) An antigen polypeptide, which is selected from the p10 regionof a BDV protein, characterized in having an ability to detect ananti-BDV antibody by using the polypeptide alone, or in combination withother antigen peptide.

[0120] 2) An antigen polypeptide characterized in that the selectedantigen polypeptide described in the above item 1) has at least 8 aminoacids.

[0121] 3) An antigen polypeptide including the amino acid sequence setout in any one of SEQ ID NOs: 5 to 8.

[0122] 4) An antigen polypeptide, which is a polypeptide that has anamino acid sequence including deletion, substitution or addition of 1 orseveral amino acids in the amino acid sequence of an antigen polypeptidedescribed in any one of the above items 1) to 3), having an ability todetect an anti-BDV antibody by using the polypeptide alone, or incombination with other antigen peptide.

[0123] The aforementioned antigen polypeptide can be selected one orseveral kinds thereof. Furthermore, it can be used in combination withother known antigen and/or antigen polypeptide. For example, it can beused in combination with an antigen polypeptide selected from the p24region and/or p40 region of a BDV protein. Specifically, it can be usedin combination with:

[0124] 1) an antigen polypeptide including an amino acid sequence setout in any one of SEQ ID NOs: 1 to 6; or

[0125] 2) an antigen polypeptide, which is a polypeptide that has anamino acid sequence including deletion, substitution or addition of 1 orseveral amino acids in the amino acid sequence described in the aboveitem 1), having an ability to detect an anti-BDV antibody by using thepeptide alone, or in combination with other antigen peptide.

[0126] In addition, a BDV antibody may be also detected using apolypeptide including one or two or more of any of the amino acidsequences set out in SEQ ID NOs: 5 to 8 in a polypeptide. Moreover, thepolypeptide set out in SEQ ID NO: 5, 6, 7 or 8 may be a polypeptide,which has an amino acid sequence including deletion, substitution oraddition of 1 or several amino acids in the original sequence thereof.

[0127] The antigen polypeptide for use in the present invention can beprepared through the synthesis according to a routine procedure, on thebasis of sequence information. Furthermore, one or multiple amino acidscan be added to the N-terminus of the antigen polypeptide as a spacerfor the purpose of allowing efficient binding of the antigen polypeptideto a solid support, a protein or the like. Although examples of theamino acid which can function as a spacer include glycine and lysine,glycine is preferred.

[0128] Examples of the sequence of the BDV antigen polypeptide, whichcan be specifically illustrated in particular, include the followings.One or two glycine shown in the leftmost part of each sequencerepresents a spacer. p24 P1:  41-55 GG-QPVDQLLKDLRKNPS (SEQ ID NO: 1)P2:  59-77 GG-DPDQRTGREQLSNDELI (SEQ ID NO: 2) p40 P3:   3-20GG-PKRRLVDDADAMEDQDLY (SEQ ID NO: 3) P4: 338-358 GG-RYRRREISRGEDGAELSGE(SEQ ID NO: 4) p10 (No/98) P5:  18-36 G-GNTTVESGRLSGGRRRSPD (SEQ ID NO:5) P6:  43-57 G-GLTKTKEDSKECTDP (SEQ ID NO: 6) p10 (Control) P7:  18-36G-GNATIESGRLPGGRRRSPD (SEQ ID NO: 7) P8:  43-57 G-GVTKTTEDPKECTDP (SEQID NO: 8)

[0129] (Sample for Detecting Antibody)

[0130] The sample for measuring an antibody of the present invention maybe any one as long as the antibody in a living body can be measured,which is not particularly limited. However, illustrative examplesinclude whole blood, plasma, serum, bonemarrow, buffycoat, urine,bodyfluid, saliva, nasal drip, lacrimal fluid, substances derived fromfaeces and the like of a human or an animal.

[0131] (Method for Detecting Antibody)

[0132] The method for detecting an antibody of the present invention isnot particularly limited, but any method can be applied as long as it isa method which can detect immunoglobulin of the IgM class alone, orconcurrently detect immunoglobulins of the IgM class and immunoglobulinsof the IgG class. In addition, the method for detecting an antibodyaccording to the present invention may be any method in which theantigen polypeptide of the present invention is used, but notparticularly limited. For this method, any one of commonly usedprocesses can be applied such as e.g., enzyme immunoassay method,fluorescent immunoassay method, radio immunoassay method, agglutinationmethod, Western blotting method, chemiluminescence immunoassay methodand the like. Exemplary particularly preferred method for the detectingincludes an immune agglutination method.

[0133] For example, measurement by a counting immunoassay method(hereinafter, referred to as “CIA method”) which is one type of theagglutination methods can be carried out by the process described inSysmex Journal Vol.20 No.1, 77-86 (1997).

[0134] Examples of supports for the sensitization with an antigenpeptide include organic polymer powder, inorganic substance powder,microorganisms, hemocytes, cell membrane pieces and the like. Examplesof the organic polymer powder which may be illustrated include insolubleagarose, cellulose, insoluble dextran and the like, and preferably,latex particles are widely utilized. Examples of the latex include e.g.,polystyrene, polystyrene-styrene sulfonate copolymer, methacrylicpolymer, acrylic polymer, acrylonitrile—butadiene styrene copolymer,vinyl chloride—acrylate ester copolymer, polyacetate vinylacrylate andthe like.

[0135] Any material and particle size of the latex particle for use isallowed as long as the latex particles form an agglutination clump uponthe antigen-antibody reaction of the immunoglobulin, without anyparticular limitation. Mean particle size of the latex is selected adlibitum depending on the detectable concentration of the subject to bemeasured or on the apparatus for the measurement to be 0.05 to 1.0 μm,preferably 0.3 to 0.85 μm. Examples of the inorganic substance powderwhich may be illustrated include silica, alumina, or metal as gold,titanium, iron, nickel or the like.

[0136] When the support for sensitization with an antigen peptide,specifically the antigen (antibody) binding latex particles, reacts withan antibody (antigen) included in a sample, the latex particlesagglutinate depending on the amount of the antibody. In the countingimmunoassay, the measurement is executed through conductingdiscrimination of the difference of the size of each particle thusagglutinated, one by one. For example, when the counted number of thenonagglutinated simple latex particle is referred to as M (Monomer); thecounted number of the agglutinated clump involving two or more latexparticles is referred to as P (Polymer); and the sum of M and P isreferred to as T (Total), degree of the agglutination can be calculatedas P/T.

[0137] For the sensitization of the antigen to the support, the antigenmay be directly sensitized to a latex particle, but any efficient methodmay be selected such as e.g., sensitization to the latex particlethrough binding of a ligand such as serum albumin or the like to theintended antigen. The amount of the antigen for the sensitization can beoptionally set as needed in the range of up to 100 μg per 1 mg of thesupport. Preferably, such an amount can be set in the range of up to 100μg in total of the aforementioned antigen peptide and/or a conjugate BDVantigen peptide in combination with a protein for use as a blockingagent for the purpose of suppressing a nonspecific reaction. Theblocking agent can be immobilized on the support after the sensitizationof the antigen, or concurrently with antigen.

[0138] According to the agglutination method including the CIA method, areaction between the antigen that is immobilized on a latex particle andthe antibody in a specimen sample leads to crosslinking among the latexparticles via the antibody to result in the formation of agglutination.In this instance, the crosslinkage can be formed whether the antibody isin the IgM class or the antibody is in the IgG class as long as areaction with the latex takes place, therefore, concurrent measurementof IgM and IgG is enabled for a specimen sample containing IgM and IgGadmixed therewith. In addition, according to the present method, theintended antibody is detected through binding with an insoluble supportsensitized with an antigen polypeptide, and thus, an additional reactionof the intended antibody is not required with an antigen, an antibody orthe like, which is additionally sensitized with a fluorescent substanceor the like. In other words, according to the present method, a BDVantibody can be similarly detected also for samples of a human and amammal other than humans, without altering the reagent. Because BDVrelates to zoonotic infections which can infect mammals such as horsesas well as humans, it is advantageous to adopt the CIA method asdemonstrated herein.

[0139] The method for the detection can be carried out in accordancewith a known method through setting the size or concentration of theinsoluble support for use, and the reaction time, by measuring theincrease or decrease of the intensity of scattering ray, the absorbanceor the intensity of transmitted beam. These methods may be used incombination of two or more types thereof.

[0140] The present invention is also directed to a kit of the reagentfor immunoassay for use in the immunoassay method as described above.Moreover, the present invention is also directed to a reagent includingan antigen polypeptide, and a reagent kit.

[EXAMPLES]

[0141] The present invention is specifically explained below withreference to Examples, however, the present invention is not anyhowlimited thereto.

(Example 1) Detection of Anti-BDV Antibody

[0142] In this Example, the presence/absence of the infection with BDVwas examined for 17 horses taken care of in Cape Toi, Miyazakiprefecture. The measurement was carried out for the serums collected in1998.

[0143] (Method for Treatment of Equine Plasma)

[0144] After the collection of blood with use of heparin, the blood wasice-cooled followed by a treatment within the day to obtain plasmasamples.

[0145] (Antigen Peptide) p24 (A) GG-QPVDQLLKDLRKNPS (SEQ ID NO: 1) p40(C) GG-PKRRLVDDADAMEDQDLY (SEQ ID NO: 3) p40 (D) GG-RYRRREISRGEDGAELSGE(SEQ ID NO: 4) p10 (G) G-GNATIESGRLPGGRRRSPD (SEQ ID NO: 7) p10 (H)G-GVTKTTEDPKECTDP (SEQ ID NO: 8)

[0146] (Sensitization of Antigen Peptide)

[0147] 1) Antigen peptide p24 (A): conjugated product prepared by adding300 μg of the peptide and 120 μg of glutaraldehyde respectively onto 24μl of 5 mg/ml bovine serum albumin (BSA) (equivalent to 120 μg of BSA)and standing the raw peptide so made for 30 minutes at 30° C.;

[0148] 2) antigen peptide p40 (C): conjugated product prepared by adding300 μg of the peptide and 120 μg of glutaraldehyde respectively onto 24μl of 5 mg/ml bovine serum albumin (BSA) (equivalent to 120 μg of BSA)and standing the raw peptide so made for 30 minutes at 30° C.;

[0149] 3) antigen peptide p40 (D): conjugated product prepared by adding300 μg of the peptide and 120 μg of glutaraldehyde respectively onto 24μl of 5 mg/ml bovine serum albumin (BSA) (equivalent to 120 μpg of BSA)and standing the raw peptide so made for 30 minutes at 30° C.;

[0150] 4) antigen peptide p10 (G): conjugated product prepared by adding300 μg of the peptide and 120 μg of glutaraldehyde respectively onto 24μl of 5 mg/ml bovine serum albumin (BSA) (equivalent to 120 μg of BSA)and standing the raw peptide so made for 30 minutes at 30° C.;

[0151] 5) antigen peptide p10 (H): conjugated product prepared by adding300 μg of the peptide and 120 pg of glutaraldehyde respectively onto 24μl of 5 mg/ml bovine serum albumin (BSA) (equivalent to 120 μg of BSA)and standing the raw peptide so made for 30 minutes at 30° C.

[0152] Whole amount of each BDV antigen conjugate as described in eachof the above 1) to 5) was added to 1 mL of a latex particle suspension(containing 5 mg of latex support having the particle size of 0.8 μm),and left to stand still at 37° C. for 1 hour. Thereafter, the latexparticles were washed to preparer the sensitized latex liquid of eachantigen. Further, 120 μg of bovine serum albumin (BSA) was similarlyadded to 1 mL of a latex particle suspension (containing 5 mg of latexsupport having the particle size of 0.8 μm), and left to stand still at37° C. for 1 hour. Thereafter, the latex particles were washed toprepare the unsensitized latex liquid.

[0153] (Detection)

[0154] Anti-BDV antibody was detected using a measuring equipmentmanufactured by Sysmex Corporation (PAMIA-50).

[0155] Onto wells of a reaction plate were added 80 μL of a buffer forthe latex agglutination reaction, 10 μL of each equine serum sample and10 μL of a solution containing the latex particles prepared as describedabove, followed by a reaction at 45° C.

[0156] After about 15 minutes elapsed following initiation of thereaction, 19 μL of the reaction mixture was added to 950 μL of Sheathfluid in the chamber of the equipment to dilute 51 times. Theagglutination reaction was terminated by the dilution, and thereafter,degree of the agglutination was detected at the optical detectionelement.

[0157] With the use of a calibration curve which had been previouslyprovided by the measurement (negative control P/T% and cut off controlP/T%), the cut off index (COI) value of the specimen was determineraccording to the formula 1. Determination was conducted as describedbelow in accordance with the COI value of the specimen.

[0158] positive: COI≧1

[0159] Negative: COI<1 $\begin{matrix}{{{Cut}\quad {Off}\quad {{Index}{\quad \quad}({COI})}} = \frac{\begin{matrix}( {{{P/T}\quad \%},{{{measured}\quad {value}\quad {of}\quad {the}\quad {specimen}} - {{P/T}\quad \%}}}  \\ {{when}\quad {unsensitized}\quad {latex}\quad {particles}\quad {were}\quad {used}} )\end{matrix}}{( {{{Cut}\quad {off}\quad {control}\quad {P/T}\quad \%} - {{Negative}\quad {control}\quad {P/T}\quad \%}} )}} & {{Formula}\quad 1}\end{matrix}$

[0160] (Results of Detection)

[0161] Results of the detection described above are presented inTable 1. Consequently, BDV positive was exhibited for all of the equineserums.

[0162] (Comparative Example 1)

[0163] Measurement was carried out for serums collected in 1998 of the17 horses described in Example 1, using an ECLIA method explained belowas a conventional method. Process for the treatment of the equine serumwas carried out similarly to that described in Example 1.

[0164] 1. Measurement Principle:

[0165] ECLIA method is based on a sandwich method in principle, in whichmicro beads bound with a BDV antigen as a solid phase and an anti-humanIgG monoclonal antibody labeled with a Ru complex that emits light uponelectrochemical alteration are employed.

[0166] Each reaction is explained below.

[0167] First reaction

[0168] Upon reaction between the antigen-bound beads and the specimen,antibodies in the specimen are bound to the antigens on the beads.

[0169] Second reaction

[0170] The antibodies bound on the beads are subjected to a reactionwith a ruthenium labeled anti-human IgG monoclonal antibody to allowbinding in a sandwiching manner.

[0171] Third reaction

[0172] When the beads are collected on an electrode and an electricenergy is imparted thereto, the Ru complex emits light depending on theamount of the ruthenium labeled anti-IgG monoclonal antibody that wasbound on the beads via the antibody. The antibody in the specimen isdetected by measuring the amount of this emitted light.

[0173] 2. Method of Detection:

[0174] Detection was performed in accordance with the followingprocedures.

[0175] (1) To a reaction tube were injected a reaction fluid 200 μL, asubject specimen 20 μL, and an antigen bound beads fluid 25 μL, and thenthe reaction was allowed at 30° C. for 9 minutes. (First reaction)

[0176] (2) A magnet was brought close to the reaction tube to collectthe beads on the wall of the reaction tube. Thereafter, the fluid in thereaction tube was aspirated to eliminate, and a washing fluid wasfurther injected followed by stirring with shaking.

[0177] (3) A magnet was brought close to the reaction tube to collectthe beads on the wall of the reaction tube. Thereafter, the fluid in thereaction tube was eliminated.

[0178] (4) To the reaction tube was injected 200 μL of a rutheniumlabeled anti-IgG monoclonal antibody fluid, and the reaction was allowedat 30° C. for 9 minutes. (Second reaction)

[0179] (5) A magnet was brought close to the reaction tube to collectthe beads on the wall of the reaction tube. Thereafter, the fluid in thereaction tube was eliminated, and a washing fluid was further injectedfollowed by stirring with shaking.

[0180] (6) A magnet was brought close to the reaction tube to collectthe beads on the wall of the reaction tube. Thereafter, the fluid in thereaction tube was eliminated, and 300 μL of a luminescent electrolytewas injected thereto. The beads were lead to a flow cell electrode tomeasure the amount of the emitted light. (Third reaction) Then,determination was conducted as positive for equal to or greater than1000, and as negative for less than 1000.

[0181]3. Antigens Used

[0182] The following two kinds of antigens were used:

[0183] BDV p24 recombinant antigen; and

[0184] BDV p40 recombinant antigen.

[0185] 4. Results

[0186] As a result, with respect to the specimens No. 1 to 23, allexhibited negative results according to the conventional method, whilstpositive results were shown for any of 5 kinds of the antigenpolypeptides according to the embodiment method, when the results of thesamples obtained in 1998 are compared (Table 1).

[0187] On the other hand, with respect to the specimens No. 24 to 36,both results of the detection according to the conventional method andaccording to the embodiment method were positive, which indicated theconsistency (Table 2). Moreover, with respect to the specimens No. 37 to47, both results of the detection according to the conventional methodand according to the embodiment method were negative, which indicatedthe consistency (Table 3).

[0188] In respect of the specimens that indicated inconsistency,positive determination was effected in the system in which the detectionwas carried out using the antigen polypeptide of the embodiment methodinvention, despite of negative results according to the conventionalmethod, thereby enabling the detection of a BDV antibody with favorablesensitivity. In regard to specimens No. 11, 12, 14, 15 and 18, the BDVantibody could be detected only with p10 (H). Accordingly, thecombination of multiple antigen polypeptides enables elevating thedetection ability of an anti-BDV antibody. TABLE 1 ConventionalEmbodiment method method (unit: C. O. I.) Positive: Number in 1998 1000or of P24 P40 P40 P10(G) P10(H) greater Specimen A: 41-55 C: 3-20 D:338-358 G: 18-36 H: 43-57 in 1998  1 2.5  1.58 0.00 0.76 4.38 228 (+)(+) (+)  2 1.3  1.02 0.26 1.4  4.14 588 (+) (+) (+) (+)  3 24.62  12.66 1.62 10.1  6.78 634 (+) (+) (+) (+) (+)  4 60.44  29.7  1.74 9.81 4.78361 (+) (+) (+) (+) (+)  5 5.12 4.7  2.36 7.12 6.18 397 (+) (+) (+) (+)(+)  6 2.88 1.14 0.02  1.140 0.36 276 (+) (+) (+)  7 1.46 3.24 0.64 1.020.60 276 (+) (+) (+)  8 11.56  6.48 0.56 5.33 2.84 116 (+) (+) (+) (+) 9 89.46  54.76  13.84  76.42  54.64   31 (+) (+) (+) (+) (+) 10 0.501.10 0.00 0.00 0.11 623 (+) 11 0.16 0.32 0.02 0.00 1.76 350 (+) 12 0.940.46 0.00 0.08 113.44  349 (+) 13 2.98 0.00 0.00 0.02 0.00 348 (+) 140.70 0.02 0.00 0.20 1.86 286 (+) 15 0.14 0.32 0.18 22.88  1.14 272 (+)(+) 16 1.08 2.50 0.00 0.10 0.00 243 (+) (+) 17 1.86 0.92 0.30 1.34 0.58230 (+) (+) 18 0.00 0.76 0.00 0.82 4.18 201 (+) 19 0.00 0.42 24.92  0.262.52 173 (+) (+) 20 0.06 0.40 3.14 0.00 0.00 108 (+) 21 0.14 3.44 0.440.18 0.18  79 (+) 22 0.88 3.72 0.34 0.38 0.00  75 (+) 23 2.12 0.08 0.240.48 0.62  48 (+)

[0189] TABLE 2 Conventional Embodiment method method (unit: C. O. I.)Positive: Number in 1998 1000 of P24 P40 P40 P10(G) P10(H) or greaterSpecimen A: 41-55 C: 3-20 D: 338-358 G: 18-36 H: 43-57 in 1998 24 0.643.4  3.44 9.11 73.92  9124 (+) (+) (+) (+) (+) 25 14.66  14.38  8.9213.30  12.06  19648  (+) (+) (+) (+) (+) (+) 26 4.02 1.64 2.44 4.89 4.0814710  (+) (+) (+) (+) (+) (+) 27 1.46 2.8  1.66 0.46 0.00 9614 (+) (+)(+) (+) 28 12.14  2.56 0.28 1.30 3.64 5960 (+) (+) (+) (+) (+) 29 1.861.62 0.40 1.64 7.02 2689 (+) (+) (+) (+) (+) 30 1.1  1.38 0.58 1.50 1.581365 (+) (+) (+) (+) (+) 31 9.36 6.7  0.00 2.16 0.10 1935 (+) (+) (+)(+) 32 0.18 0.24 28.24  0.68 33.84  1642 (+) (+) (+) 33 0.12 0.04 0.000.24 7.24 2930 (+) (+) 34 2.78 1.38 0.46 0.60 0.32 1813 (+) (+) (+) 354.84 0.74 0.22 1.16 0.76 1799 (+) (+) (+) 36 0.42 3.62 23.42  0.00 0.001703 (+) (+) (+)

[0190] TABLE 3 Conventional Embodiment method method (unit: C. O. I.)Positive: Number in 1998 1000 of P24 P40 P40 P10(G) P10(H) or greaterSpecimen A: 41-55 C: 3-20 D: 338-358 G: 18-36 H: 43-57 in 1998 37 0.000.00 0.00 0.00 0.00 407 38 0.56 0.22 0.16 0.00 0.00 103 39 0.58 0.340.20 0.64 0.14 165 40 0.12 0.24 0.00 0.00 0.00 449 41 0.00 0.00 0.000.00 0.00 373 42 0.04 0.06 0.00 0.12 0.00  76 43 0.00 0.08 0.00 0.000.00 443 44 0.02 0.00 0.52 0.00 0.00 138 45 0.00 0.72 0.32 0.00 0.00 27646 0.62 0.80 0.08 0.28 0.00 263 47 0.54 0.00 0.00 0.12 0.00 352

[0191] (Experimental Example 1)

[0192] Ratio of IgM and IgG in the detected antibodies was determinedfor specimens No. 5 and 6 which indicated inconsistent results of thedetection according to the conventional method and to the embodimentmethod, as well as specimens No. 25 and 26 which indicated consistentresults of the detection.

[0193] Anti-equine IgM goat serum (COSMO BIO Co., Ltd.) and anti-equineIgG goat serum (COSMO BIO Co., Ltd.) stock solutions were employed asthe reagent for absorption. A mixture of a buffer and an anti-equine IgMserum at a ratio of 9:1 was used as an IgM absorption buffer, andsimilarly, a mixture of a buffer and an anti-equine IgG serum at a ratioof 9:1 was used as an IgG absorption buffer.

[0194] Measurement of P/T% was carried out for the IgM absorption bufferand the IgG absorption buffer with PAMIA-50 in a similar manner to thatdescribed in Example 1. Rate of IgM absorption and rate of IgGabsorption were determined in accordance with the formula 2.$\begin{matrix}{{{Rate}\quad {of}\quad {IgM}\quad {absorption}} = {\frac{\begin{matrix}{( {{COI}\quad {of}\quad {the}\quad {specimen}} ) -} \\( {{COI}\quad {in}\quad {the}\quad {IgM}\quad {absorption}\quad {buffer}} )\end{matrix}}{{COI}\quad {of}\quad {the}\quad {specimen}} \times 100\%}} & \lbrack {{Formula}\quad 2} \rbrack \\{{{Rate}\quad {of}\quad {IgG}\quad {absorption}} = {\frac{\begin{matrix}{( {{COI}\quad {of}\quad {the}\quad {specimen}} ) -} \\( {{COI}\quad {in}\quad {the}\quad {IgG}\quad {absorption}\quad {buffer}} )\end{matrix}}{{COI}\quad {of}\quad {the}\quad {specimen}} \times 100\%}} & \quad\end{matrix}$

[0195] Results of the detection of serums collected in 1998, 1999 and2001 of the specimen No. 5 are shown in FIGS. 1 to 3, respectively;results of the detection of serums collected in 1998 to 2001 of thespecimen No. 6 are shown in FIGS. 4 to 7, respectively; results of thedetection of serum collected in 1998 of the specimen No. 25 are shown inFIG. 8; and results of the detection of a serum collected in 1998 of thespecimen No. 26 are shown in FIGS. 9.

[0196] Consequently, with respect to specimens No. 5 and No. 6 withdifference found among the results of the detection according to theconventional method and the present method for the detection, the moreappearance of IgM was found than the appearance of IgG in serum samplescollected in 1998 and 1999, while no appearance of IgG was found in aserum sample collected in 2001. On the other hand, with respect tospecimens No. 25 and No. 26 with no difference found among the resultsof the detection according to the conventional method and the embodimentmethod, the more appearance of IgG was found than the appearance of IgMin a serum sample collected in 1998.

[0197] From these results of experiment, it was revealed that a longperiod of time was required for the class switching from IgM to IgGfollowing the infection with BDV, with respect to specimens 5 and 6.

(Example 2)

[0198] For 82 horses taken care of in Cape Toi, Miyazaki prefecture,survival rate in 5 years was studied for the group determined as beingBDV antibody-positive and negative using the antigen polypeptide of theembodiment method according to each method for the detection, i.e., as aresult of the detection according to the method described in Examples 1,and as a result of the detection according to the method of ComparativeExample that is a conventional method of a serum collected in 1998.Consequently, as shown in Table 4, mortality rate in the instance ofantibody positive was 66.7%, whilst mortality rate in the instance ofantibody negative was 28.3%, when the detection was carried outaccording to the embodiment method. On the other hand, when thedetection was carried out according to the conventional method,mortality rate in the instance of antibody positive was 52.4%, however,mortality rate of 42.6% was presented even in the instance of antibodynegative. When the measurement was carried out using the antigenpolypeptide of the present invention, high mortality rate was exhibitedin the instance of antibody positive, thus, the results of themeasurement reflected the mortality rate. Therefore, to conduct moreaccurate examination of BDV infection was permitted. TABLE 4 Follow upof BDV antibody positive horses 1998 2002 Agglutination Positive 3612(33.3%) confirmed as alive method  7(19.4%) confirmed as dead(Embodiment 17(47.2%) uncertain death + uncertain = method) 24(66.7%)Agglutination Negative 46 33(71.7%) confirmed as alive method 4(8.7%)confirmed as dead (Embodiment  9(19.6%) Uncertain death + uncertain =method) 13(28.3%) Conventional Positive 21 10(47.6%) Confirmed as alivemethod  5(23.8%) confirmed as dead  6(28.6%) uncertain death + uncertain= 11(52.4%) Conventional Negative 61 35(57.4%) confirmed as alive method6(9.8%) confirmed as dead 20(32.8%) uncertain death + uncertain =26(42.6%)

[0199] [Industrial Applicability]

[0200] According to the method for the detection in which the antigenpolypeptide of the present invention is used, a sample, which had notbeen able to be detected when the measurement according to theconventional method was carried out, could be also determined as BDVpositive, as a result of the concurrent measurement of IgM and IgG forBDV. The present invention enables the determination of thepresence/absence of an infection through detecting the appearance of IgMwithout overlooking, also in instances of exogenous antigens such asBDV, which necessitate a long period of time for the class switchingfrom IgM to IgG of the immunoglobulins, where the antigen itself can notbe detected owing to the slight amount of the antigen.

[0201] Additionally, the demonstrated CIA method herein is extremelyuseful as a method of examining BDV, because BDV which may be the causeof zoonotic infections can be detected using the identical reagent forhorses, humans and the like.

1 8 1 15 PRT Borna disease virus 1 Gln Pro Val Asp Gln Leu Leu Lys AspLeu Arg Lys Asn Pro Ser 1 5 10 15 2 17 PRT Borna disease virus 2 Asp ProAsp Gln Arg Thr Gly Arg Glu Gln Leu Ser Asn Asp Glu Leu 1 5 10 15 Ile 318 PRT Borna disease virus 3 Pro Lys Arg Arg Leu Val Asp Asp Ala Asp AlaMet Glu Asp Gln Asp 1 5 10 15 Leu Tyr 4 19 PRT Borna disease virus 4 ArgTyr Arg Arg Arg Glu Ile Ser Arg Gly Glu Asp Gly Ala Glu Leu 1 5 10 15Ser Gly Glu 5 19 PRT Borna disease virus 5 Gly Asn Thr Thr Val Glu SerGly Arg Leu Ser Gly Gly Arg Arg Arg 1 5 10 15 Ser Pro Asp 6 15 PRT Bornadisease virus 6 Gly Leu Thr Lys Thr Lys Glu Asp Ser Lys Glu Cys Thr AspPro 1 5 10 15 7 19 PRT Borna disease virus 7 Gly Asn Ala Thr Ile Glu SerGly Arg Leu Pro Gly Gly Arg Arg Arg 1 5 10 15 Ser Pro Asp 8 15 PRT Bornadisease virus 8 Gly Val Thr Lys Thr Thr Glu Asp Pro Lys Glu Cys Thr AspPro 1 5 10 15

What is claimed is:
 1. A method for detecting an antibody wherein anexamination of a disease caused by an exogenous antigen is conducted,said exogenous antigen being an antigen having a property in which theclass switching from the IgM antibody to the IgG antibody ofimmunoglobulin antibodies raised against said antigen is achieved aftertwo months following the appearance of the IgM antibody, said methodcomprising detecting the IgM antibody to said exogenous antigen.
 2. Themethod for detecting an antibody according to claim 1 wherein thedetection of the antibody to said exogenous antigen is carried out bydetecting the IgM antibody alone, or concurrently detecting the IgMantibody and IgG antibody.
 3. The method for detecting an antibodyaccording to claim 1 wherein said exogenous antigen is a microorganism,virus and/or proteinous substance which may be the cause of a disease ofa human or of a mammal other than humans.
 4. The method for detecting anantibody according to claim 1 wherein said exogenous antigen is Bornadisease virus (BDV).
 5. A method for detecting an antibody wherein anexamination of a disease caused by Borna disease virus (BDV) isconducted, said method comprising detecting the IgM antibody alone, orconcurrently detecting the IgM antibody and IgG antibody which is(are)raised against the virus.
 6. The method for detecting an antibodyaccording to claim 1 wherein said method for detecting an antibody is animmune agglutination reaction method.
 7. The method for detecting anantibody according to claim 6 wherein said immune agglutination reactionmethod is a fine particle counting immunoassay method.
 8. A reagent fordetecting an anti-BDV antibody which has an antigen polypeptide selectedfrom the p10 region of a Borna disease virus (BDV) protein.
 9. Thereagent for detecting an anti-BDV antibody wherein the antigenpolypeptide according to claim 8 comprises an antigen polypeptide has atleast 8 amino acids.
 10. The reagent for detecting an anti-BDV antibodywherein the antigen polypeptide according to claim 8 has a polypeptidewhich includes an amino acid sequence set out in SEQ ID NO: 5, 6, 7 or8.
 11. A method for detecting an anti-BDV antibody wherein the antigenpolypeptide according to claim 8 is used.
 12. A method for detecting ananti-BDV antibody wherein the antigen polypeptide according to claim 8,and an antigen polypeptide selected from the p24 region and/or p40region of a Borna disease virus (BDV) protein are used.
 13. The methodfor detecting an anti-BDV antibody wherein the antigen polypeptide fromthe p24 region according to claim 12 has a polypeptide including anamino acid sequence set out in SEQ ID NO: 1 or
 2. 14. The method fordetecting an anti-BDV antibody wherein the antigen polypeptide from thep40 region according to claim 12 has of a polypeptide including an aminoacid sequence set out in SEQ ID NO: 3 or
 4. 15. The method for detectingan anti-BDV antibody according to claim 11 wherein said method fordetecting an anti-BDV antibody is an immune agglutination reactionmethod.
 16. The method for detecting an antibody according to claim 15wherein said immune agglutination reaction method is a fine particlecounting immunoassay method.